The role of Plasmodium V-ATPase in vacuolar physiology and antimalarial drug uptake

Significance Malaria is caused by Plasmodium parasites, which replicate within human erythrocytes. During intraerythrocytic growth, the parasite takes up and digests host cell hemoglobin in an acidified vacuole. Several antimalarials interfere with biochemical pathways in this vacuole leading to parasite death. It was long believed that accumulation of these drugs is a mere function of the pH gradient between parasite cytosol and the vacuolar lumen. By targeting the multimeric proton pump powering this gradient, we identify distinct functions of this protein complex in physiology and maintenance of the digestive vacuole. Contrary to current belief, we find that severe dissipation of the proton gradient has only limited impact on antimalarial drug uptake and no effect on drug susceptibility.


Supporting text -Methods
Generation and validation of transgenic parasites P. falciparum B11 parasites constitutively expressing DiCre were co-transfected with a pDC2 guide plasmid inducing Cas9-mediated double strand cleavage of the locus of interest, together with a linearized repair template (1). Templates were generated by commercial gene synthesis and contained 5′ and 3′ homology regions and a recodonized sequence of the gene. In case of the conditional knockout mutants, two loxP sites were introduced into the sequence, one within an artificial intron and one downstream of the stop codon. Tagging with fluorescent proteins was achieved by cloning mCh, mNG or SEP into the repair template in frame with the coding sequence, using HpaI and EcoRV restriction sites. Transgenic parasites were selected with WR99210 and cloned by limiting dilution. Isolation of transgenic parasite clones and DiCre-mediated genomic excision were confirmed by diagnostic PCR and fluorescence microscopy. Primers for molecular cloning, guide plasmid construction and diagnostic PCR as well as repair template sequences are included in Fig. 1B and SI Appendix, Table S1 and Sequences S1 to 13. Transgenic parasite strains generated in this study are listed in SI Appendix, Table S2.

Drug sensitivity assays
Drug sensitivity assays were performed as described previously (2). In brief, synchronous B cKO parasite cultures were seeded into 96 well plates 20 hours post invasion at a final volume of 200 μl per well at 2% hematocrit and 1% parasitemia in the presence of RAP (20 nM) or DMSO, respectively. Antimalarial compounds at varying concentrations were added in triplicate 20 or 36 hours post invasion. Plates were incubated for a total of 72 hours to allow for parasite reinvasion to occur. Following cell lysis, parasite DNA was labelled with 1x SYBR Gold stain (Invitrogen) and fluorescence was quantified using an EnVision multilabel plate reader (PerkinElmer, 485 nm excitation, 535 nm emission. Uninfected RBCs served as a background control (for raw data, see Dataset S1). For drug interaction studies, CQ was mixed with the V-ATPase inhibitors at fixed ratios of 5:0, 4:1, 3:2, 2:3, 1:4, and 0:5. The drug combinations were then diluted and used in drug sensitivity assays as described above. Mean ∑FIC values were calculated and interpreted as described previously (3,4).

Quantitative live fluorescence microscopy
For quantification of protein expression, DV autofluorescence, vacuolar morphology and V-ATPase subunit dissociation, parasites were recorded 42 hours post invasion. For quantification of hemerelated autofluorescence, the red signal of the DV of non-tagged parasites was captured using a standard Texas Red filter cube configuration. Vacuolar fluorescence intensity and area were determined after manual outlining of the DV. To determine levels of V-ATPase subunit expression, the signal was captured from the entire parasite. For all intensity measurements, a background correction was performed. Dissociation of V-ATPase subunits from the DVM was analyzed by generating a fluorescence intensity profile along a transect spanning the DV lumen and the parasite cytoplasm. A fragmented DV was indicated by the presence of two or more autonomous compartments positive for vacuolar markers. Hemozoin was quantified by polarization microscopy as described previously (5). Stainings with MitoTracker Red CMXRos and Lysosensor Blue DND-167 (Thermo Fisher Scientific) were performed according the manufacturer's instructions. The pH sensor was calibrated by incubating pm2-sep schizonts for 30 minutes in media of varying pH supplemented with 50 µM of nigericin, followed by quantitative live fluorescence microscopy. For time-lapse microscopy, c cKO + crt-mNG parasites were seeded onto concanavalin A-coated microscopy dishes and imaged at 37°C in intervals of 30 minutes on a temperature-controlled FV1000 confocal microscope (Olympus) (6).
Microscopic quantification of Fluo-CQ fluorescence in live parasites was performed as described previously (7). Parasites at schizont stage protected from light were stained with 500 nM Fluo-CQ at 37°C for 30 minutes in the presence or absence of 61 nM ConA and washed twice in complete culture medium before being adjusted to a hematocrit of ~50%. 1.5 µl of the cell suspension were transferred onto a glass slide, covered with a cover slip (24 x 65 cm) and immediately imaged over a period of 5 to 7 minutes. Parasites were first identified by DIC using minimal illumination to avoid photobleaching of the probe and were then imaged using 460-500 nm excitation and 512-542 nm emission filters, which overlap well with the spectrum of the fluorescent nitrobenzofurazan moiety of Fluo-CQ (excitation/emission maxima at 467/539 nm). To ensure comparability and identical degrees of photobleaching for all samples, we used a consistent exposure time that did not cause oversaturation of the images. The area of the DV was outlined manually and the fluorescence was measured as background-corrected raw integrated density. Non-specific signal was controlled for by imaging unstained samples. The selected Fluo-CQ concentration and incubation time were optimized to achieve conditions which do not saturate the uptake or the overall capacity of the DV for this probe. This was indicated by our observation that intravacuolar Fluo-CQ fluorescence was linearly correlated with external probe concentration and incubation time at this range (SI Appendix, Fig. S11).

Cytosolic pH approximation with BCECF-AM
For cytosolic pH measurements, DMSO18h and RAP18h-treated B cKO parasites at schizont stage were released from their host cells by saponin lysis (0.05% in complete culture medium) and washed three times with HEPES-buffered saline (120 mM NaCl; 5 mM KCl, 25 mM HEPES; 20 mM glucose, 1 mM MgCl2; pH 7.1). Washed parasites were stained with the acetoxymethyl ester of BCECF (5 µM; Invitrogen) for 20 minutes at 37°C and washed five times for 5 minutes each in HEPES-buffered saline. Then, parasites were resuspended in 200 μl HEPES-buffered saline and 10 μl of this suspension were added to 490 µl of complete medium adjusted to varying pH values containing either 50 µM nigericin or vehicle only. Following a 20-minute incubation at room temperature, parasite suspensions were transferred to black flat-bottom 96 well plates at a volume of 150 µl per well. Throughout the entire staining procedure, parasites were protected from light to avoid photobleaching of BCECF. Using an EnVision multilabel plate reader (PerkinElmer), samples were excited at wavelengths of 440 and 490 nm and fluorescence was detected at 535 nm. Unstained parasites in complete culture medium served as a background control. The ratio of both background-corrected fluorescence values (490/440) was calculated and cytosolic pH values were inferred from the calibration curves obtained from nigericin-treated parasites.

Quantification of intracellular [ 3 H]CQ accumulation
Briefly, schizont-infected RBCs were magnet-purified and resuspended in 1 ml of prewarmed reaction buffer A (bicarbonate-free RPMI 1640 supplemented with 11 mM glucose, 25 mM HEPES and 2 mM glutamine; pH 7.3) containing 40 nM [ 3 H]CQ (20 Ci/mmol; American Radiolabeled Chemicals) in combination with 61 nM ConA or vehicle at a hematocrit of 25,000 to 30,000 cells per µl, as determined with a Z1-Coulter Particle Counter (Beckman Coulter). Cells were incubated at 37°C and 75 µl aliquots were removed in duplicate at the indicated time points and diluted with an equal volume of ice-cold reaction buffer A. For efflux experiments, infected RBCs were incubated for 15 minutes in reaction buffer A containing 20 nM [ 3 H]CQ. After preloading, cells were washed twice in ice-cold medium and resuspended in prewarmed reaction buffer A containing no or 61 nM ConA. The reaction was held at 37°C and duplicate 150 µl aliquots were removed from the reaction at various time points.
Aliquots from the CQ response assays were spun through a 5:4 mixture of dibutyl phthalate and dioctyl phthalate, and the aqueous phase containing unincorporated [ 3 H]CQ was removed and transferred to a scintillation vial. The cell pellets were recovered by cutting the reaction tubes through the oil layer with a sharp scalpel while squeezing the tubes with tweezers. The tips of the tubes containing the cell pellets were placed in fresh reaction tubes and incubated with 66 µl ethanol and 33 µl NCS tissue solubilizer (Amersham Biosciences) overnight at 55°C. Lysates were then acidified and bleached by the addition of 25 µl 1 N HCl and 25 µl 30% H2O2 and transferred to a scintillation vial. Radioactivity of the aqueous phases and the cell lysates was measured using a Tri-carb 2100TR liquid scintillation counter (Packard). The intracellular drug concentration was calculated from the amount of radio-labeled drug taken up by the cells and by assuming a volume of 75 fl for an infected RBC (8). Drug accumulation was expressed as the ratio of intracellular versus extracellular drug concentration.